synthesize the necessary constituents by using environmental conditions
and
maintain their lives by performing their chemical and physical processes under the influence of extracellular chemical and physical factors
to sustain their lives.
They provide the continuation of their strain through reproduction.
Reproduction = Increase in cell count
In clinical microbiology laboratories, the isolation and identification of pathogenic microorganisms for the diagnosis of infectious diseases is very important.
Microorganisms must be able to be produced under suitable conditions for conducting researchs in medical microbiology, pharmaceutical microbiology, food technology, industrial microbiology, environmental microbiology and many other fields.
Because of differentiation of enzyme systems
the physical and chemical conditions that microorganisms need for nutrition
and reproduction differ.
In terms of energy source;
Phototroph (using light energy)
Chemotroph (using chemical substance)
In terms of carbon source;
Autotroph (organic matter synthesis from inorganic substances)
Heterotroph (using ready organic material)
In terms of hydrogen source;
Lithotroph (using inorganic substances) and Organotroph (using organic substances)
Pathogens?
Heat
Psychrophilic bacteria -8 / +15 Mesophilic bacteria +20 / +45 Thermophilic bacteria +50 / +70
Osmotic pressure
Optimal osmotic pressure High osmotic pressure Low osmotic pressure
Carbon source
Hydrogen donors and receivers Nitrogen source
Oxidation / reduction potential
pH
Most microorganisms are in the range of pH 6-8 (neutral pH) Some microorganisms such as yeasts and molds are in acidic conditions
Some microorganisms such as V. chlorea are in alkaline conditions…
Oxygen
Aerobes
Anaerobes
Facultative anaerobes Microaerophiles
CO2
Minerals
Growth factors and vitamins Water
Microorganisms can be produced in-vitro or in-vivo under suitable environmental conditions in laboratory.
Microorganisms such as viruses, ricketsia, chlamydia that can survive only in living cells.
Most of the bacteria and fungi can be growth in non-living environments.
Experimental animals Embryonated eggs Cell cultures
Tissue cultures
Media
A nutrient blend that microorganism can be produced in vitro and contains materials necessary for the production are called as medium.
There is no common optimum medium formula for all microorganisms.
According to physical
characteristics Liquid
Semi-solid Solid
According to usage purposes
Simple (basal) Complex
Spesific Differential Selective
Both selective and differential
Enrichment Preservation Transfer
According to chemical
structures Natural Semi-
synthetic Synthetic
It is derived from some red seaweed species.
It is thickener.
The ratio of agarose / agaropectin in it determines the consistency.
It is not a food source for microorganism.
Dissolves above 90 °C, solidifies below 45 °C.
It does not affect the pH, but at low pH it hardens the solidification of the medium.
It has a water holding capacity of 300-500 times.
Agar free
Nutrient Broth
Mueller Hinton Broth
Agar ratio is between 0.3 - 0.5 %
Stuart transport media
Kirschner semi-solid media
Agar ratio is between 1.5 – 3 %
Nutrient Agar
Mueller Hinton Agar
Liquid
Semi Solid
Solid
There are also biphasic media that carry both solid and liquid phases in the same medium.
Castenada medium
Natural media
Contains natural substances such as bouillon, peptone, milk, egg, blood, serum, potatoe.
Their chemical content is undefined.
Synthetic media
Medium containing pure chemical substances.
It is a well-known medium containing defined pure chemical substances at certain ratios.
Semi-synthetic media
It contains both chemical substance and various organic substances.
The content is not exactly known.
Routinely used in laboratories
Contain sufficient amounts of nutrients for the development of many microorganisms
Do not contain inhibitory
substances
Simple (basal) media
Basic media containing essential nutrients such as peptone water, bouillon
peptone water peptone + salt + water Bouillon peptone + salt + bouillon
gelose bouillon + agar
Usage areas; first isolation, counting,
production.
Growth inducing enriched media can be obtained by adding more nutrients such as blood, serum, egg to the basic medium.
blood agar, chocolate agar, tomato juice agar
Complex media
It is used for purposes such as production and identification of microorganisms, obtaining pure culture, examination of physico-chemical
activities.
Indicators may contain some special
substances that induce or inhibit the germination.
Complex media
a) Spesific media: Used for the production of only one species. Spesific to species.
Löwenstein Jensen media Mycobacterium tuberculosis
Complex media
b) Selective media: The medium prepared with
the substances that allow the growth of the desired microorganism while inhibiting the growth of unwanted groups of microorganisms from a mixed culture.
This feature is provided by dyes, antibiotics, etc.
SS Agar Salmonella and Shigella spp.
Selenite F Salmonella spp
Complex media
c) Differential media: It is the medium which shows its metabolism end products and whether the microorganism uses a specific nutrient.
It contains various indicator.
Endo Agar Lactose (+) and Lactose (-) Enterobacteriaceae differentiation
Complex media
d) Both selective and differential nutrients:
Selectivity by allowing the growth of a group of microorganisms and inhibiting the growth of the other group; as well as by differentiating the biochemical characterization of the microorganisms.
d) Both selective and differential nutrients:
Eosin Methylen Blue Agar Allows the generation of Gram (-) bacteria. Different E. coli and Klebsiella spp.
Complex media
e) Reagent media: It is the medium which enables the formation of various reactions
according to the biochemical character of the microorganism.
Results interpreted by the colour change
Simmons Citrate Agar media the use of citrate as a carbon source Clark-Lubs media the presence of (+) or (-) glucose fermentation
Complex media
f) Enrichment media: If the number of desired microorganisms in the mixed culture is low, it is the medium which multiplies it by the addition of various substances and causes the others to reproduce in a lesser amount.
Selenite F media Salmonella spp.
Alkaline-peptonic water Vibrio chlorea
Complex media
g) Storage medium: It is the medium used to keep the isolated microorganisms in the refrigerator at -20 ºC or -80 ºC for a long time. For example;
Certain proportions of glycerol or fat-free milk.
Complex media
h) Transfer media: The medium from which the samples are carried until they are transferred to the laboratory and cultured.
Cultural appearance may be homogeneous turbidity, granular reproduction, bottom depression
membrane form on the surface for the liquid medium and
colony shape for solid medium.
The size, shape, colour, flavour, structure, appearance of the colony varies depending on the type of bacteria.
S (smooth) type colony:
R (rough) type colony:
M (mucoid) type colony:
L type colony:
Production of microorganisms culturing Produced microorganisms culture
One type of microorganism culture pure culture Sample inoculated to the medium inoculum
Culturing to medium inoculation
Apart from the microorganisms present in the culture medium or in the medium, other microorganisms called as contaminants, this state is called as contamination.
Bacterial growth rate during reproduction (generation time) depends on the;
species of bacteria and ambient conditions
When a certain number of microorganisms are added to a liquid medium and incubated;
with sampling at regular intervals, it is observed that there are different periods of the generation depending on the time.
Log of numbers of bacteria
Time
1. Latent period:
Adjustment period
No multiplication, even some of them die
Enlarge the volume, synthesize enzymes and intermediate metabolites
The duration depends on the type, number, age of the microorganism
2. The period when the generation accelerated:
Period of the beginning of the generation Cell size is at its maximum size
3. Logarithmic reproduction period:
Exponential reproduction period as soon as possible
Food is reduced, toxic substances start to form Reduced amount of oxygen in aerobic conditions Multiplication speed is reduced
5. The period of proliferation:
Proliferation continues
Bacterial death keeps the number of living ones in balance
6. Reduction period of bacteria:
The number of dead cells is greater than the number of cells divided
7. Logarithmic reduction period:
Conditions become very inappropriate Rapid death starts
Logarithmic reduction in count
8. Re-arrangement period:
Number is miserable
Low number of deaths and generation
Survivors can stay alive for weeks or months, depending on their genus
Sporadic species form spores
1. Amoeboid Motion: It's seen in amoeba. The microorganism moves with the help of pseudopods.
2. Slip Motion: Some blue-green algae and algae-like bacteria, make a sliding motion by curling ,with their twisting bodies in moist solid media.
3. Spiral Motion: Microorganisms whose bodies are spirally twisted act as auger with the help of axial filaments on moist media.
4. Colony Motion: The colonies of some microorganisms are displaced by a very slow sliding movement in the moist solid medium.
5. Flagellar Motion: Microorganisms with flagella are replaced by this movement. It is an active motion. For this movement there is a need for energy that is provided from ATP.
(taxis)
6. Brownian Motion: Bacteria that do not have movement organelles can cause vibration, rotation, bending, swing, etc.
in the environment without changing their place. It is called a passive motion, which is caused by the collision of microorganisms with other molecules in the liquid medium.
Hanging Drop Slide inspection
Wet Mount Slide inspection
(Inspection between slide-cover glass)
Inspection by Soft Agar Stabbing (Inspection in Semi-Solid Media)
Inspection on Solid Medium
Motility test of microorganisms Wet mount slide
Brownian motion Flagellar motion
• Klinik mikrobiyolojik Tanı. Prof.Dr. Hakkı Bilgehan. 1992, 680 p.
• Principles of Microbiology, 2th edth. Ronald M. Atlas.1997, 1298 p.
• Temel Mikrobiyoloji ve Bağışıklık Bilimi, Prof.Dr.Hakkı Bilgehan, 2000.
• Mikrobiyoloji Pratik Kitabı, Editör A. Tevfik Cengiz, 2001.
